CN116643036B - RNA modified living cell imaging system and composition and application thereof - Google Patents
RNA modified living cell imaging system and composition and application thereofInfo
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Abstract
本发明属于生物分子技术领域，具体涉及一种RNA修饰的活细胞成像系统及其组合物和应用。该RNA修饰的活细胞成像系统包含RNA报告体系和FRET探针，其中，RNA报告体系包括四环素诱导表达体系、mMaroon1荧光蛋白、NLS‑细胞核定位序列和22个重复的m6A‑MS2报告序列；FRET成像探针包含YTH结构域、YPet荧光蛋白、长链柔性连接臂、ECFP荧光蛋白、MCP蛋白和NLS核定位信号。本专利开发的m6A修饰的活细胞成像系统可在活细胞内进行m6A修饰的高分辨实时动态成像，可实现m6A修饰水平和RNA定位与翻译的三重动态监控，从而搭建m6A修饰通路的研究平台和药筛平台。
The present invention belongs to the field of biomolecular technology, and in particular relates to a living cell imaging system for RNA modification, and its composition and application. The RNA-modified living cell imaging system comprises an RNA reporter system and a FRET probe, wherein the RNA reporter system comprises a tetracycline-inducible expression system, an mMaroon1 fluorescent protein, an NLS-nuclear localization sequence, and a 22-repeat m6A-MS2 reporter sequence; the FRET imaging probe comprises a YTH domain, a YPet fluorescent protein, a long-chain flexible linker, an ECFP fluorescent protein, an MCP protein, and an NLS nuclear localization signal. The m6A-modified living cell imaging system developed in this patent can perform high-resolution real-time dynamic imaging of m6A modification in living cells, and can achieve triple dynamic monitoring of m6A modification levels and RNA localization and translation, thereby building a research platform and drug screening platform for m6A modification pathways.
Description
Technical Field
The invention belongs to the technical field of biological molecules, and particularly relates to an RNA modified living cell imaging system, a composition and application thereof.
Background
RNA epigenetic modification is a key process to regulate expression of posttranscriptional genes. N6-A methylation modification (m 6A) is the most abundant known form of RNA modification and is widely involved in regulating RNA metabolism, three-dimensional genomic structure and DNA damage modification. About 50% of the RNA has 4 and less m6A modifications, and the modified sequences share the common feature of RRACH (r=a/G; h=a/U/C). The m6A modification of RNA is mainly dynamically regulated by methylase METTL/14 complex and demethylases FTO and ALKBH5, and is realized by recognition and function of binding proteins YTHDF/2/3, YTHDC/2 and IGF2BP 1/2/3. modulation of RNA fate by m6A modification is a hotspot and difficulty in the current basic research. Previous studies have suggested that m6A modification is involved in regulating the transcription, transport, degradation and translation processes of RNA, but specific mechanisms have not yet established consensus.
A number of related proteins of the m6A modified pathway were demonstrated for carcinomatous effects. METTL3 is demonstrated to be abnormally elevated in acute myelogenous leukemia (acute myeloid leukemia, AML) and is closely related to the occurrence and progression of AML. METTL 3's specific inhibitor STM2457 is effective in inhibiting proliferation of human AML tissue in a mouse model. Furthermore, the level of demethylase ALKBH5 was shown to be inversely related to glioblastoma clinical efficacy. Detailed studies indicate that alk bh5 stabilizes FOXM1 by removing the m6A modification of the cancer marker FOXM1, promoting its translation, and thus promoting canceration. Furthermore, recent studies have found that the m6A modification and its recognition protein YTHDF2 are enriched in Amyotrophic Lateral Sclerosis (ALS) patients, and that knocking down YTHDF2 can reduce TDP43 and C9ORF72 mediated neurotoxicity. Furthermore, more than 90% of TDP43 binding RNAs were confirmed to have m6A modification. Therefore, development of small molecule modulators against related proteins of the m6A modified pathway, such as METTL/14, FTO, ALKBH5, and the like, is of great importance.
Aiming at the dilemma and difficulty of the current m6A modification research, the development of m6A modification living cell imaging means with space-time precision is an important breakthrough point for revealing the physiological and pathological functions of m6A modification, and is also a vacuum zone of the related research of m6A modification. The existing m6A modification detection means mainly relies on the m6A antibody to carry out chromogenic imaging on a fixed cell sample, and dynamic observation and construction of a living cell medicine sieve platform are difficult to realize.
The invention patent with publication number CN110567906A discloses a method for characterizing RNA methylation modification, which is characterized in that a theoretical calculation method is applied to obtain characteristic absorption spectrums of an RNA sequence and an RNA m6A methylation sequence aiming at an RNA m6A methylation modification path, a characterization method for dynamic modification of RNA methylation based on the spectrum method is established, and the invention does not relate to an RNA modified living cell imaging means.
Disclosure of Invention
In view of the above, the invention innovatively constructs an m6A modified RNA report system and a FRET imaging system, and can perform m6A modified high-resolution real-time dynamic imaging in living cells.
It is an object of the present invention to provide a composition for use in the preparation of RNA-modified living cell imaging systems.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
A composition for preparing an RNA-modified living cell imaging system, the composition comprising an RNA reporter comprising an inducible expression system, a protein tag, a subcellular organelle localization sequence, an RNA epigenetic modification sequence, and/or an RNA aptamer sequence, and a FRET probe comprising an RNA epigenetic modification binding domain, a FRET pair, and/or an RNA aptamer binding protein.
Further, in the RNA reporting system, the induction expression system is a tetracycline induction expression system, the protein tag is mCherry fluorescent protein, CFP fluorescent protein, GFP fluorescent protein, mMaroon fluorescent protein, halo tag, SNAP tag and/or luciferase, the subcellular organelle localization sequence is SKL-peroxisome localization sequence, NLS-nuclear localization sequence and/or NES-nucleation localization sequence, and the RNA epigenetic modification sequence and the RNA aptamer sequence are 22 repeated m6A-MS2 reporting sequences and/or PP7-BoxB sequences.
Further, in the FRET probe, the RNA epigenetic modification binding domain is an YTH domain, the FRET pair is YPET fluorescent protein and ECFP fluorescent protein, GFP fluorescent protein and mCherry fluorescent protein and/or BFP fluorescent protein and GFP fluorescent protein, and the RNA aptamer binding protein is MCP protein, PCP protein and/or N22 protein.
Further, the RNA reporter system further comprises a connecting sequence, and the FRET probe further comprises a structural sequence and a connecting sequence.
It is a second object of the present invention to provide an RNA-modified living cell imaging system.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
an RNA-modified living cell imaging system comprising a subject one of said compositions.
Further, the RNA modifications include modifications of different nucleotide types, in particular adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide and uracil ribonucleotide;
Further, the RNA modification includes methylation modification, acetylation modification, phosphorylation modification, hydroxymethylation modification, and/or 2' -O-methylation modification.
Further, the RNA modification includes an m6A modification, an m4C modification, an m1A modification, an m5C modification, an hm5C modification, an m7G, a 2' -O-methylation modification, an ac4C acetylation modification, and/or an m3U modification.
At present, no literature on RNA m6A modified live cell imaging technology has been retrieved. Known detection means for m6A modifications mainly include in vitro tube experiments or most common immunofluorescence. It was found by search that the imaging of specific sequence m6A modifications was achieved by the person skilled in the art by employing antibody detection and rolling circle amplification techniques on fixed cell samples, however this method is difficult to achieve dynamic observations and to construct a viable cell drug screen platform.
Further, the m6A modified RNA reporting system consists of a tetracycline induction expression system with a nucleotide sequence shown as SEQ ID NO.1, mMaroon fluorescent proteins with a nucleotide sequence shown as SEQ ID NO.2, NLS-nuclear localization sequences with a nucleotide sequence shown as SEQ ID NO.3 and 22 repeated m6A-MS2 reporting sequences with a nucleotide sequence shown as SEQ ID NO. 4.
Further, the PCR primer for mMaroon fluorescent protein amplification comprises a forward primer mMaroon F1 and a reverse primer mMarron R1, the nucleotide sequence of mMaroon F1 is shown as SEQ ID NO.5, and the nucleotide sequence of mMarron R1 is shown as SEQ ID NO. 6.
Further, the nucleotide sequence of the RNA report system is shown as SEQ ID NO. 7.
Further, the m6A modified FRET probe consists of an YTH domain with a nucleotide sequence shown as SEQ ID NO.8, a YPET fluorescent protein with a nucleotide sequence shown as SEQ ID NO.9, a long-chain flexible connecting arm with a nucleotide sequence shown as SEQ ID NO.10, an ECFP fluorescent protein with a nucleotide sequence shown as SEQ ID NO.11, an MCP protein with a nucleotide sequence shown as SEQ ID NO.12 and an NLS nuclear localization signal with a nucleotide sequence shown as SEQ ID NO. 13.
Further, the nucleotide sequence of the FRET probe is shown as SEQ ID NO. 14.
It is a further object of the present invention to provide a stable cell line obtained by transfecting the living cell imaging system.
It is a fourth object of the present invention to provide a method for detecting the level of RNA modification using the living cell imaging system.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
A method for detecting the RNA modification level by using the living cell imaging system comprises the steps of coexpression of a reporter RNA and a FRET probe in mammalian cells in vitro, and detection of the RNA modification level by measuring the FRET/ECFP ratio.
When the RNA reporter system is not modified by m6A, the long-chain flexible connecting arm effectively separates YPET and ECFP proteins, thereby reducing FRET background signals. When an m6A modification occurs, MCP binds to the MS2 aptamer, the YTH domain binds to the adjacent m6A modification, pulling YPet closer to ECFP, enhancing FRET signal.
It is a fifth object of the present invention to provide the use of a combination of said RNA reporter and said FRET probe for dynamic monitoring of m6A modification levels, RNA localization and translation in living cells.
Further, the living cells are mammalian living cells cultured in vitro.
It is a sixth object of the present invention to provide the use of said composition, said living cell imaging system and/or said stably transformed cell line for dynamically monitoring the level of m6A modification, RNA localization and translation in living cells.
Further, the living cells are mammalian living cells cultured in vitro.
Further, the composition and/or the living cell imaging system are used in research platforms and drug screening platforms for constructing RNA modification pathways.
Still further, the RNA modification is an m6A modification, an m4C modification, and/or an m3U modification.
Further, the use of said composition and/or said RNA-modified living cell imaging system in the screening of living cell drugs for modification related enzymes.
Still further, the related enzymes are methylase METTL/14 complex, demethylase FTO and/or albh 5.
The methods of the invention encompass all reporter systems and FRET probes that employ similar design principles for epigenetic modification of RNA (including, but not limited to, m6A modification). Specifically, the reporter system includes the use of an inducible expression system other than tetracycline, fluorescent proteins or other tag proteins other than mMaroon, other subcellular organelle localization sequences other than NLS, m6A modification motif or other RNA epigenetic modification sequences other than GGACC, other RNA aptamer sequences other than MS2, and linker sequences other than those shown in the patent. In the FRET probes, there are included FRET pairs that employ an m6A recognition domain and other RNA epigenetic modification binding domains that differ from YTH, RNA aptamer binding proteins that differ from MCP, and different structural order and linking sequences. The application of this patent is not limited to the research platform and drug screening platform mentioned in this patent for constructing m6A modified pathways. It will be understood by those skilled in the art that various modifications and equivalent substitutions may be made to the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention, and it is intended to cover the scope of the claims of the present invention.
The invention has the beneficial effects that:
1. the main innovation point of the patent is that an m6A modified report system is constructed, the system has the function of inducing expression, and simultaneously has an original m6A modified indication sequence of 22X, wherein the m6A modified motif is connected with an MS2 aptamer, and meanwhile, the patent constructs an m6A modified FRET probe for the first time and is used for realizing high-resolution dynamic tracing in living cells;
2. The m6A modified living cell imaging system developed by the patent comprises an m6A modified RNA report system and a FRET imaging system, can perform m6A modified high-resolution real-time dynamic imaging in living cells, performs dynamic observation, and has wider detection dimension;
3. The three-layer dynamic monitoring of RNA positioning, m6A modification level and RNA translation can be realized by combining the m6A modified living cell report RNA system and the FRET probe, so that a research platform and a medicine sieve platform of an m6A modified passage are built;
4. The design concept of the patent can be used for developing other RNA modified living cell imaging systems, researching physiological and pathological functions of the living cell imaging systems and developing medicaments;
5. The m6A modified living cell imaging system developed by the patent does not depend on an m6A antibody, and the cost is lower;
6. The m6A modified living cell imaging system developed by the patent can construct a stable cell line, and is easy to construct a living cell medicine sieve platform of m6A modified related enzyme.
Drawings
FIG. 1 is a schematic illustration of the detection principle of an m6A modified living cell imaging system;
FIG. 2 is a graph showing the results of expression of the m6A modified FRET probe in mammalian cells;
FIG. 3 is a graph of the results of an m6A modified living cell imaging system for specifically detecting the level of m6A modification;
FIG. 4 is a graph showing the results of dynamic detection of live cells by wild-type probe (WT BS) on the m6A reporter;
FIG. 5 is a graph showing the results of dynamic detection of live cells by a Mutant probe (Mutant BS) on an m6A reporter system.
Detailed Description
The technical scheme of the present invention will be further clearly and completely described in connection with specific embodiments. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. Therefore, all other embodiments obtained by those skilled in the art without undue burden are within the scope of the invention based on the embodiments of the present invention.
In the application, the following components are added:
DOX, doxycycline, is a tetracycline derivative;
22X m6A-MS2 sequences, 22 repeated m6A modification motif and MS2 aptamer combination report sequences;
mMaroon1 fluorescent protein is a red fluorescent dye widely used in biotechnology as a tracer, and comprises molecular markers, cell component positioning and the like;
NLS nuclear localization signals are a type of polypeptide sequence that can assist in the transport of marker proteins into the nucleus;
YTH domain, which is a protein domain that specifically binds to m6A modified RNA, is widely present in m6A modified binding proteins;
YPET is the yellow fluorescent protein with the strongest brightness and has good light stability;
a long-chain flexible connecting arm is a connecting sequence commonly used for constructing a FRET probe in a molecule, and the sequence mainly comprises glycine and serine, has very high flexibility and relatively large length, and can reduce the background of FRET;
ECFP fluorescent protein, enhanced cyan fluorescent protein;
MCP protein is a binding protein of MS2 aptamer, and can bind the MS2 aptamer with high specificity and binding force;
EXAMPLE 1 live cell imaging method of m6A modification
The method comprises the following steps:
1) Fusing a tetracycline induction expression system, mMaroon fluorescent proteins, NLS positioning sequences and 22 repeated m6A modified report sequences to obtain m6A modified report RNA plasmids;
2) Fusion YTH structural domain, YPET fluorescent protein, long-chain flexible connecting arm, ECFP fluorescent protein, MCP protein and NLS nuclear localization signal to obtain m6A modified FRET probe plasmid;
3) Fusion of YTH structural domain mutants, YPET fluorescent proteins, long-chain flexible connecting arms, ECFP fluorescent proteins, MCP proteins and NLS nuclear positioning signals to obtain a function deletion plasmid of the m6A modified FRET probe;
4) In mammalian cells, the reporter RNA and FRET probe are co-expressed and the effect of the FRET/ECFP ratio on tetracycline induction is observed.
In the step 1), the nucleotide sequence of the tetracycline-inducible expression system is shown as SEQ ID NO.1, the nucleotide sequence of mMaroon fluorescent protein is shown as SEQ ID NO.2, the nucleotide sequence of NLS positioning sequence is shown as SEQ ID NO.3, and the nucleotide sequence of m6A-MS2 report sequence is shown as SEQ ID NO. 4.
In the step 2), the nucleotide sequence of YTH structural domain is shown as SEQ ID NO.8, the nucleotide sequence of YPET fluorescent protein is shown as SEQ ID NO.9, the nucleotide sequence of long-chain flexible connecting arm is shown as SEQ ID NO.10, the nucleotide sequence of ECFP fluorescent protein is shown as SEQ ID NO.11, the nucleotide sequence of MCP protein is shown as SEQ ID NO.12, and the nucleotide sequence of NLS nuclear localization signal is shown as SEQ ID NO. 13.
The specific principle and flow are shown in figure 1. The core of the patent lies in the design of an m6A modified RNA reporter system and a FRET imaging probe. Wherein, the RNA report system is a section of artificially designed and assembled gene sequence, and a tetracycline induction expression system, mMaroon fluorescent proteins, NLS-nuclear localization sequences and 22 repeated m6A-MS2 report sequences are spliced by a molecular biological technology. The reporter is combined with rtTA-VP64 under the induction of tetracycline or DOX to initiate transcription of RNA. After RNA transcription, the 22X m6A-MS2 sequence recruits METTL/14 methylase complex to make m6A modifications to specific sites. After the m6A modified RNA is transported to cytoplasm, the RNA is combined with ribosome to translate into mMaroon red fluorescent protein which has NLS-nuclear localization sequence and can guide mMaroon1 to be localized in cell nucleus. In another aspect, the FRET imaging probe comprises an YTH domain, a YPet fluorescent protein, a long chain flexible linker arm, an ECFP fluorescent protein, an MCP protein, and an NLS nuclear localization signal. Wherein the YTH domain can recognize the m6A modification of RNA, YPET and ECFP are FRET pairs, and have higher FRET conversion efficiency, and MCP protein can bind MS2 aptamer.
When the FRET probe is present with the m6A modified reporter RNA, the YTH domain binds to its m6A modification site and the MCP protein binds to the adjacent MS2 sequence, thereby pulling the spatial distance of YPET and ECFP, enhancing the FRET signal. Conversely, if the YTH domain is mutated, the m6A modification site cannot be bound, and YPet and ECFP form a lower FRET background due to the mutual principle of the long-chain flexible linker arm spacing.
Example 2 plasmid construction and evaluation of reporter RNA System
The sequence of 22X m6A-MS2 was designed, and a gene fragment of 22X m6A-MS2 was obtained using custom gene synthesis services and inserted into pUC Kan+ plasmid. pUC Kan+ plasmid was digested with BamH1 and KpN.sup.1, and the target fragment containing 22X m6A-MS2 was recovered. mMaroon1 sequences were amplified from pmMaroon1 (non-commercial plasmid) using mMaroon F1 and mMaroon R1 primers and the amplified sequences were digested with Nhe1 and KpN1 to recover the fragment of interest comprising mMaroon 1. The pLD1112 plasmid was digested with Nhe1 and BamH1, and then the ends were treated with CIP enzyme to recover the vector sequence. The pLD1112 vector, mMaroon1 and 22X m6A-MS2 were ligated using T4 ligase, 16 degree ligation overnight. The ligation product is transfected to Stbl3 competence, a single clone is selected on a coated plate, a sanger sequencing is adopted to identify and select correct plasmids, and finally pLD1112NLS-mMaroon1-NLS-22X m6A-MS2 plasmids, hereinafter referred to as reporter RNA plasmids, the nucleotide sequence of which is shown as SEQ ID NO.7 are obtained. RNA plasmids were transiently reported in HeLa cells using lipo3000 transfection kit. 36 hours after transfection, expression of mMaroon1 was induced by adding 1. Mu.g/mL DOX, and the fluorescence intensity of mMaroon1 was compared before DOX induction and after 12 hours of induction. After 12 hours of DOX induction, mMaroon1 had significantly increased fluorescence intensity.
MMaroon1 amplified PCR primer mMaroon F1 has nucleotide sequence shown in SEQ ID NO.5, mMaroon R1 has nucleotide sequence shown in SEQ ID NO.2, and amplified nucleotide sequence shown in SEQ ID NO. 6.
Example 3 construction of wild-type m6A modified FRET Probe plasmid
Searching and comparing the gene sequence of the YTH domain, obtaining the gene fragment of the YTH domain by adopting a custom-made foundation synthesis service, and inserting the gene fragment into pUC Kan+ plasmid. YTH F and YTH R primers were used to amplify YTH sequences from pUC Kan+. YPet-linker-ECFP sequences were amplified from pSin H K9Me3 FRET BS (non-commercial plasmid) using YPet F and ECFP R. MCP-NLS sequences were amplified from addgene 75383 (commercial plasmid) using MCP F and MCP R6. The vector sequence was recovered by cleavage pSin H K9Me3 FRET BS with Nhe1 and EcoR1 followed by treatment of the ends with CIP enzyme. By usingHiFi DNAAssembly ligation pSin vector, YTH, YPET-linker-ECFP and MCP-NLS,50℃for 1 hour. The ligation product was transfected into DH 5. Alpha. Competent, the coated plate was picked up for monoclonal, sanger sequencing was used to identify and select the correct plasmid, and finally the wild m6AFRET probe plasmid, hereinafter referred to as WT BS plasmid, was obtained, the nucleotide sequence of which was shown as SEQ ID NO. 14.
The nucleotide sequence of the YTH F PCR primer is shown as SEQ ID NO.15, and the nucleotide sequence of the YTH R PCR primer is shown as SEQ ID NO. 16.
The nucleotide sequence of YPET F and ECFP amplified PCR primer is shown as SEQ ID NO.17 and the nucleotide sequence of ECFP R is shown as SEQ ID NO. 18.
The nucleotide sequence of the MCP-NLS amplified PCR primer is shown as SEQ ID NO.19, and the nucleotide sequence of the MCP R6 is shown as SEQ ID NO. 20.
EXAMPLE 4 construction of mutant m 6A-modified FRET Probe plasmid
Searching and comparing the gene sequence of the mutant YTH domain, obtaining the gene fragment of the YTH domain by adopting a custom-made foundation synthesis service, and inserting the gene fragment into pUC Kan+ plasmid. The mutant YTH sequences were amplified from pUC Kan+ using YTH F and YTH R primers. Other fragments were consistent with example 3. By usingHiFi DNAAssembly ligation pSin vector, mutant YTH, YPET-linker-ECFP and MCP-NLS,50 degree ligation for 1 hour. The ligation product is transfected to DH5 alpha competence, a single clone is picked up by plating, a correct plasmid is identified and selected by sanger sequencing, and finally a Mutant m6A FRET probe plasmid, hereinafter referred to as a Mutant BS plasmid, is obtained. The reporter plasmid was transiently transformed in Hela cells using lipo3000 transfection kit. The expression of the probe was observed 36 hours after transfection, and the results of the specific experiments are shown in FIG. 2. Both WT BS and Mutant BS are mainly expressed in cytoplasm, and WT BS forms a small amount of granular structure, which is not the case with Mutant BS.
EXAMPLE 5 feasibility and specificity evaluation of m6A modified living cell imaging System
RNA plasmids and WT BS or Mutant BS plasmids were transiently reported in HeLa cells using lipo3000 transfection kit. After 36 hours of transfection, hela cells were induced with DOX. After induction for 5 hours, fluorescence imaging pictures of ECFP, YPet, FRET and mMaroon1 were taken using a rotary disk confocal microscope with FRET function, and the difference in FRET/ECFP fluorescence intensity ratio in WT BS or in the instant BS group was analyzed. The experimental results are shown in FIG. 3, where WT BS and reporter RNA plasmid co-expressed, cells exhibited higher FRET/ECFP ratios. In addition, fluorescence intensities of ECFP, YPet, FRET and mMaroon1 were monitored in real time using a time series imaging mode, and it was found that the ratio of mMaroon to FRET/ECFP increased gradually over time after DOX induction in the WT BS group, and the increase in the ratio of FRET/ECFP was earlier than mMaroon1. In the Mutant BS group, only the increase of mMaroon1 fluorescence intensity was observed, and the FRET/ECFP fluorescence intensity ratio was not significantly changed, and the specific experimental results are shown in FIG. 4 and FIG. 5.
Finally, it should be noted that the above implementation is only for illustrating the technical solution of the present invention and is not limiting. The methods of the invention encompass all reporter systems and FRET probes that employ similar design principles for epigenetic modification of RNA (including, but not limited to, m6A modification). Specifically, the reporter system includes the use of an inducible expression system other than tetracycline, fluorescent proteins or other tag proteins other than mMaroon, other subcellular organelle localization sequences other than NLS, m6A modification motif or other RNA epigenetic modification sequences other than GGACC, other RNA aptamer sequences other than MS2, and linker sequences other than those shown in the patent. In the FRET probes, there are included FRET pairs that employ an m6A recognition domain and other RNA epigenetic modification binding domains that are different from YTH, RNA aptamer binding proteins that are different from MCP, and different structural order and linking sequences that are not used for YPET/ECFP. The application of this patent is not limited to the research platform and drug screening platform mentioned in this patent for constructing m6A modified pathways. It will be understood by those skilled in the art that various modifications and equivalent substitutions may be made to the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention, and it is intended to cover the scope of the claims of the present invention.
Claims (4)
1. The composition for preparing the m6A modified living cell imaging system is characterized by comprising an m6A modified RNA reporter system and an m6A modified FRET probe, wherein the m6A modified RNA reporter system comprises a tetracycline-inducible expression system, mMaroon fluorescent proteins, NLS-nuclear localization sequences and 22 repeated m6A-MS2 reporter sequences, the nucleotide sequences of the m6A modified FRET probe are shown as SEQ ID NO.7, and the m6A modified FRET probe comprises YTH domains, YPET fluorescent proteins, long-chain flexible connecting arms, ECFP fluorescent proteins, MCP proteins and NLS nuclear localization signals, and the nucleotide sequences of the m6A modified FRET probe are shown as SEQ ID NO. 14.
A m6a modified living cell imaging system comprising the composition of claim 1.
3. Use of the composition of claim 1 and/or the living cell imaging system of claim 2 for dynamically monitoring the level of m6A modification, RNA localization and translation in living cells.
4. Use of the composition of claim 1 and/or the living cell imaging system of claim 2 for screening living cell drugs for modification of a related enzyme.
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